Exploring the potential of bis(thiazol-5-yl)phenylmethane derivatives as novel candidates against genetically defined multidrug-resistant Staphylococcus aureus

Antimicrobial resistance (AMR) represents an alarming global challenge to public health. Infections caused by multidrug-resistant Staphylococcus aureus (S. aureus) pose an emerging global threat. Therefore, it is crucial to develop novel compounds with promising antimicrobial activity against S. aureus especially those with challenging resistance mechanisms and biofilm formation. Series of bis(thiazol-5-yl)phenylmethane derivatives were evaluated against drug-resistant Gram-positive bacteria. The screening revealed an S. aureus-selective mechanism of bis(thiazol-5-yl)phenylmethane derivatives (MIC 2–64 μg/mL), while significantly lower activity was observed with vancomycin-resistant Enterococcus faecalis (MIC 64 μg/mL) (p<0.05). The most active phenylmethane-based (p-tolyl) derivative, 23a, containing nitro and dimethylamine substituents, and the naphthalene-based derivative, 28b, harboring fluorine and nitro substituents, exhibited strong, near MIC bactericidal activity against S. aureus with genetically defined resistance phenotypes such as MSSA, MRSA, and VRSA and their biofilms. The in silico modeling revealed that most promising compounds 23a and 28b were predicted to bind S. aureus MurC ligase. The 23a and 28b formed bonds with MurC residues at binding site, specifically Ser12 and Arg375, indicating consequential interactions essential for complex stability. The in vitro antimicrobial activity of compound 28b was not affected by the addition of 50% serum. Finally, all tested bis(thiazol-5-yl)phenylmethane derivatives showed favorable cytotoxicity profiles in A549 and THP-1-derived macrophage models. These results demonstrated that bis(thiazol-5-yl)phenylmethane derivatives 23a and 28b could be potentially explored as scaffolds for the development of novel candidates targeting drug-resistant S. aureus. Further studies are also warranted to understand in vivo safety, efficacy, and pharmacological bioavailability of bis(thiazol-5-yl)phenylmethane derivatives.


Introduction
Antimicrobial resistance (AMR) represents an alarming global challenge to public health.The escalating emergence of resistance, particularly against last-line antibiotics, poses a significant threat, leading to increased mortality rates and escalating treatment costs [1][2][3].Annually, an estimated 17 million individuals succumb to bacterial infections, with over 2 million individuals are affected by drug-resistant strains [4].The rapid spread of AMR underscores the urgent need for comprehensive and novel strategies targeting drug-resistance among clinically important pathogens.
The need for novel antimicrobial agents with unique mechanisms of action is particularly evident in the case of Gram-positive pathogens, especially multidrug-resistant (MDR) Staphylococcus aureus (S. aureus) [5,6].This becomes more evident in infections caused by methicillin-resistant S. aureus (MRSA) or vancomycin-resistant (VRSA) S. aureus, particularly those cases with surgically implanted medical devices like prostheses or catheters, which require assertive treatment strategies often involving surgical removal [7,8].Additionally, the ability of S. aureus to form biofilms is associated with prolonged and persistent infections, demanding extended treatment durations [8,9].Hence, there is a growing need for novel antimicrobial compounds exhibiting activity that effectively targets both bacteria and their biofilms [10].
Infections caused by S. aureus exhibit considerable heterogeneity in their nature, necessitating a multifaceted treatment approach that encompasses the administration of staphylococciactive antibiotics alongside surgical debridement, removal of indwelling devices or aggressive wound management [11][12][13][14].Several antibiotics with established clinical efficacy are deployed to combat staphylococcal infections, targeting crucial bacterial processes including cell wall biosynthesis, transcription, translation, and DNA replication [10,11,14].Resistance to antibiotics in S. aureus emerges through various mechanisms, including modification of the drug target, enzymatic inactivation of the drug, enhanced drug efflux, and the dissemination of mobile genetic elements that facilitate resistance development [14,15].Notably, the rapidly growing antibiotic resistance poses an increasing challenge, particularly with respect to vancomycin, a crucial antibiotic employed in the treatment of drug-resistant staphylococcal infections [15].Although the occurrence of VRSA or vancomycin-intermediate S. aureus (VISA) strains remains relatively rare, its prevalence is on the rise [15].Consequently, it is crucial to develop novel compounds capable of combatting S. aureus strains harboring diverse pre-existing resistance mechanisms.
Thiazole, a five-membered heterocyclic ring containing both sulfur and nitrogen, has emerged as a versatile scaffold in the development of various antimicrobial drugs [16].Thiazole inherent chemical properties, such as reactive and electron-rich structure, lend themselves well to various substitutions, allowing the generation of diverse derivatives with enhanced bioactivity.Moreover, thiazole derivatives, often has moderate lipophilicity and the potential for hydrogen bonding, contribute to favorable pharmacokinetic profiles [16,17].Thiazole derivatives have displayed efficacy against a various pathogenic organism, including WHO priority listed pathogens [16,17].Depending on the structural substitutions, these compounds often target essential microbial processes such as DNA synthesis, protein translation, and direct and indirect cell wall biosynthesis [16,18].The sulfur atom within the thiazole ring can coordinate with metal ions crucial for microbial enzyme activity, adding another layer to their antimicrobial mechanism [18,32].The thiazole nucleus containing compound shows favorable biological properties with the ability to interact with diverse microbial targets including cell wall synthesis pathway, thus making thiazole-based compounds as a promising scaffold for the development of novel and effective antimicrobial agents for further pre-clinical evaluation against multidrug-resistant S. aureus or other Gram-positive pathogens [11,14,17,32].
In our previous paper, we described the initial synthesis of high molecular weight bis(thiazol-5-yl)phenylmethane derivatives [18,19].These compounds demonstrated structuredepended antimicrobial activity against Mycobacterium luteus (M.luteus).As a continuation of our interest to develop novel bioactive compounds targeting multidrug-resistant pathogens, these compounds were screened using drug-resistant bacterial and fungal pathogens.In the current study, we describe the subsequent characterization of bis(thiazol-5-yl)phenylmethane as promising candidates selectively targeting S. aureus even with the presence of challenging drug-resistance mechanisms.The molecular in silico modeling studies reveled that most potent bis(thiazol-5-yl)phenylmethane derivatives 23a and 28b interacts with active center of S. aureus MurC protein, thus acting as novel modulator of MurC in S. aureus.Finally, the most promising hit compounds were able to induce the dispersion of mature S. aureus biofilms, thus making bis(thiazol-5-yl)phenylmethane as promising scaffold for further development of S. aureus-directed antimicrobial candidates.

General procedure for the preparation of thiazoles 7, 8b
To a solution of compound Ib (0.7 g, 2.5 mmol) in acetone (10 mL), the corresponding haloketone (3.1 mmol) was added, and the mixture was refluxed for 2 h.Upon completion of the reaction (TLC), the mixture was cooled down, and the formed aminothiazolium bromide was filtered off, washed with acetone, dried and transferred into the base by refluxing in 2.5% aqueous sodium acetate (0.5 g/20 mL), cooling and filtering off the formed solid to give compound 7b as a blueish solid, m. p. 168−169˚C and compound 8b as white solid, m. p. 188-189˚C.

General procedure for the preparation of bis(thiazol-5-yl)phenylmethanes (24-31)b.
To a mixture of the corresponding compound (6-9)b (3 mmol), the appropriate aromatic aldehyde (1.5 mmol) (molar ratio 2:1) and acetone (40 mL), the concentrated hydrochloric acid (0.5 mL) was added dropwise.The mixture was heated at reflux for 18 h and cooled down.The formed crystalline product was filtered off, washed with plenty of acetone and boiled in 4% aqueous sodium acetate for 5 min.The obtained appropriate product (24-31)b was filtered off, washed with water and dried.

Bacterial strains and culture conditions
The multidrug-resistant S. aureus strain TCH 1516 [USA 300-HOU-MR] was obtained from the American Type Culture Collection (ATCC

Minimal inhibitory concentration determination
The antimicrobial activity of bis(thiazol-5-yl)phenylmethane derivatives was assessed using the broth microdilution method, following the guidelines outlined by the Clinical Laboratory Standards Institute (CLSI), with modification [20].In brief, the compounds were dissolved in dimethylsulfoxide (DMSO) to attain a final concentration of 25 mg/mL.The control antibiotics and antifungals were obtained from MCE (MedChemExpress, United States) and dissolved in DMSO.Dilution series were prepared in deep 96-well microplates to achieve a two-fold concentration range of 0.25, 0.5, 1, 2, 4, 8, and 16 μg/mL, utilizing Cation-Adjusted Mueller-Hinton Broth (CAMHB) as the growth medium.For C. difficile experiments, the dilutions were performed in CAMBH supplemented with 10% of leaked horse blood and 100 μg/mL of L-cysteine.For C. auris screening, dilutions were performed in RMPI/MOPS media.The microplates containing the dilution series were then inoculated with fresh cultures of each tested organism to reach a final concentration of 5 × 10 4 CFU (colony-forming units) of the test organism in media containing 1% DMSO and 1× drug concentration, with a volume of 200 μl per well.Wells that were inoculated with media containing 1% DMSO served as positive controls.Subsequently, the microplates were incubated at 35 ± 1˚C for 18 ± 2 hours.For C. difficile experiments, microplates were sealed in commercial GasPack anaerobic system and incubated at 35 ± 1˚C for 24 hours.Following the incubation period, the plates were examined using a manual microplate viewer (Sensititre Manual Viewbox, United States).The minimal inhibitory concentration (MIC) was defined as the lowest concentration (μg/mL) of the tested drug that completely inhibited the growth of the test organism.All experiments were conducted in duplicate with three technical replicates for each condition.

Minimal bactericidal concentration determination
The minimal bactericidal concentration (MBC; μg/mL) of bis(thiazol-5-yl)phenylmethane derivatives was determined as described before with slight modification [20].Following the MIC determination using 96-well plates, an aliquot (10μL) from MIC well and wells with increasing concentrations were taken and spotted on Columbia Sheep Blood agar.The plates were incubated at 35 ± 1˚C for 24 hours and the MBC concentration was determined as the lowest concentration (μg/mL) of test compounds that is fully suppressing the growth of test organism.All experiments were conducted in duplicate with three technical replicates for each condition.

Biofilm integrity assay
Bacterial biofilm integrity assay was performed as described elsewhere with minor modifications [20].Briefly, the test organism was cultured on Mueller-Hinton agar overnight to achieve well isolated colonies.One-two colonies were picked up and suspended in 5 mL of CAMBH and cultured overnight.The bacterial cultures were then diluted 1:100 with fresh CAMBH and subsequently cultured at 37˚C, 200 rpm for 3-4 hours until OD600 reached 0.3.The culture was then dispensed in 96-well flat-bottomed microplates and was cultured overnight in static conditions to generate the mature biofilms.Next day, the media was aspirated and replaced with fresh CAMBH, containing 0.5% of DMSO and selected test compounds.The biofilms were further incubated for 24 hours at 37˚C at static conditions.After cultivation, media was aspirated, the wells were washed 3 times with sterile PBS solution and fixed with 4% paraformaldehyde for 30 min, at room temperature.The paraformaldehyde was aspirated, fixed biofilms washed 3 times with PBS and stained with 0.5% of crystal violet for 1 hour.The plates were washed in running deionized water, dried at room temperature.The biofilm absorbed crystal violet was then solubilized with 10% aques acetic acid for 10 min, transferred to fresh flat bottom microplates and the absorbance was measured using a Multiscan Sky microplate spectrophotometer at OD.

Molecular docking
Ligand preparation.The 3D structure of each compound (23a and 28b) was built using Gaussview and was geometrically optimized using Avogadro [21].These structures were visually checked to correct some structural errors.
Docking of ligand-protein interaction.We resorted to virtual screening using Autodock Vina, a target-specific scoring method useful for virtual screening [21][22][23].The derivatives 23a and 28b were docked into a set of proteins to identify potential biological targets.Both ligands and proteins were prepared using AutoDock Tools version 1.5.7 (ADT) according to the Auto-Dock Vina High Throughput screening standard method.Gasteiger partial charges were assigned to the atoms of ligands.The AutoTors option was used to define the rotatable bonds in the ligands.The visual inspection of the results was performed using the Molecular Graphics Laboratory (MGL) Tools package.We selected a grid volume enough to cover each receptor.Finally, graphical analysis was performed using Discovery Studio Visualizer, version 1.9.2 and Discovery Studio.
Human THP-1 monocytes (ATCC TIB-202), also obtained from ATCC, were maintained in complete RPMI medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with GlutaMAX (ThermoFisher Scientific, Waltham, MA, USA), 10% heat-inactivated FBS (Gibco, ThermoFisher Scientific, Waltham, MA, USA), and 50 μM of beta-mercaptoethanol.Both cell lines were cultivated at 37˚C under 5% CO 2 atmospheric conditions.For the initiation of differentiation of THP-1 monocytes into macrophages, the cells were exposed to complete RPMI medium containing 200 ng/mL of Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Schnelldorf, Germany) for a duration of 48 hours.Subsequently, the cells underwent a resting period in PMA-free RPMI medium for an additional 48 hours prior to their utilization in experimental assays.
Cytotoxicity assay.The in vitro inhibitory effects of the compounds were assessed using the MTT assay, as described in previous studies [24,25].The A549 or THP-1 derived macrophages were seeded into 96-well plates at a concentration of 1 × 10 4 cells per well.Following an overnight incubation at 37˚C with 5% CO 2 , the cells were treated with the compounds at a concentration of 100 μM, and this treatment was carried out in triplicate.After a 20-hour exposure period, the MTT reagent was introduced, and the cells were subsequently incubated for an additional 4 hours.To serve as a cytotoxicity control, a 10% solution of sodium dodecyl sulfate (SDS) was employed.The formazan product resulting from the MTT assay was extracted using anhydrous dimethyl sulfoxide (DMSO).The optical density of the samples was determined using a microplate reader, specifically at a wavelength of 570 nm.To ascertain the percentage of A549 cell viability, the following formula was applied: ([AE-AB]/[AC-AB]) × 100%, with AE representing the absorbance of the experimental samples, AC representing the absorbance of untreated samples, and AB representing the absorbance of blank controls.The obtained data were subjected to analysis using statistical software such as GraphPad Prism or QuickCalcs.
Rabbit erythrocyte hemolysis assay.Rabbit erythrocytes were suspended in prewarmed phosphate buffered saline (PBS) containing 0.1% of DMSO.Then test compounds were added to reach 100 μM concentration and then samples were incubated for 1 hour at 37˚C.10% SDS was used as a hemolysis control.Following the incubation, samples were centrifuged at 1000 × g for 10 min., and the released hemoglobin was measured spectrophotometrically at the optical density of 405 nm (OD 405 nm).

Statistical analysis.
The results are expressed as mean ± standard deviation (SD).Statistical analyses were performed with Prism (GraphPad Software, San Diego, CA, USA), using Kruskal-Wallis test and two-way ANOVA.P < 0.05 was accepted as significant.

Synthesis of compounds
The interest to the synthesis and investigation of bis(thiazoles) (10-23)a and (24-31)b follows from the fact that analogous compounds exhibited a promising antibacterial activity against M. luteum in our previously publication [19].Therefore, in this study we have explored the synthetic versatility and structure-depended activity (SAR) of bis(thiazoles) against panel of multidrug-resistant Gram-positive and Gram-negative pathogens with emerging multidrugresistance phenotypes.

Enhanced antimicrobial activity against Gram-positive pathogens by bis (thiazol-5-yl)phenylmethane derivatives (10-23)a and (24-31)b
To comprehensively evaluate the antimicrobial properties of bis(thiazol-5-yl)phenylmethane derivatives 10-31, we conducted antimicrobial activity determination assay using a panel of representative multidrug-resistant bacterial and fungal strains.The antimicrobial activity of these compounds was first evaluated at a fixed concentration of 100 μM.We refined the CLSI broth dilution method and employed resazurin viability assays in combination with spectrophotometric analysis to thoroughly characterize the inhibitory potential of compounds 10-31 against the WHO priority bacterial and fungal strains.

Bis(thiazol-5-yl)phenylmethane derivatives 10-31 exhibit limited antimicrobial activity against drug-resistant Enterococcus, Streptococcus, and Clostridioides difficile
After observing antimicrobial activity directed against S. aureus TCH 1516 by compounds 10-31, we sought to investigate whether the observed efficacy of bis(thiazol-5-yl)phenylmethane derivatives 10-31 is specific to S. aureus or if it extends to other Gram-positive bacterial pathogens.To address this question, we selected representative Gram-positive strains of clinical significance and determined their minimal inhibitory concentrations (MIC; μg/mL) following the guidelines set by the CLSI (Table 2).
Furthermore, naphthalene derivatives (27-29)b containing a nitro radical at the R 2 position demonstrated antimicrobial activity against C. difficile.Compound 27b (R 1 -H) exhibited weak activity, with an MIC of 64 μg/mL, while the replacement of R 1 -H with fluorine (28b) had no effect.Intriguingly, the introduction of a bromine atom at the R 1 position (29b) significantly increased the activity against C. difficile, resulting in an MIC of 16 μg/mL.These findings underscore the importance of substituents at the R 1 position for directing antimicrobial activity against anaerobic C. difficile.

Bis(thiazol-5-yl)phenylmethane derivatives 10-31 exhibit promising activity against Staphylococcus aureus strains with emerging resistance mechanisms
After characterizing the antimicrobial activity of compounds 10-31 against a panel of Grampositive clinical strains, including Enterococcus spp., Streptococcus spp., and Clostridiodes spp., we sought to determine whether the observed antimicrobial activity against S. aureus was influenced by pre-existing antimicrobial resistance phenotypes.To address this question comprehensively, we selected representative strains with varying resistance profiles, encompassing methicillin-susceptible (MSSA) wild-type, MRSA, and VRSA (Table 3).
Our investigations revealed that compounds 10-31 exhibited a structure-dependent antimicrobial activity, which was only slightly affected by the pre-existing resistance profiles of the S. aureus strains.Both MSSA, MRSA, and VRSA strains displayed similar susceptibility profiles, with MIC values ranging from 2 to 64 μg/mL.Notably, compounds containing a p-tolyl core with a fluorine atom substitution at the R 2 position demonstrated structure-dependent activity when the R 1 position was modified with halogen or a nitro group (Table 3).
The introduction of a nitrile group at the R 1 position and a chlorine atom at the R 2 position resulted in equal antimicrobial activity against all resistance types, with a MIC of 8 μg/mL observed for compound 13a.However, replacing the nitrile at the R 1 position with a nitro substituent 14a decreased antimicrobial activity against MRSA and VRSA strains, leading to an MIC of 16 μg/mL although increased activity was observed against pan-susceptible S. aureus SA-1001 (MSSA).Subsequently, substituting chlorine with bromine at the R 2 position in the nitro compound 14a resulted in compound 15a.Compound 15a demonstrated favorable antimicrobial activity against S. aureus strains with emerging resistance phenotypes, including VRSA.Furthermore, compound 15a which exhibited similar MIC values across all S. aureus MSSA (8-16 μg/mL) and MRSA (16-32 μg/mL) strains, except for S. aureus TCH 1516 MRSA strain where MIC was observed as 32 μg/mL (Table 3).
The importance of the R 2 position in the antimicrobial activity of bis(thiazol-5-yl)phenylmethanes was further elucidated by introducing an acidic electron-withdrawing nitro group at the R 2 position and varying substituents at the R 1 position.Compound 16a with R 1 -H displayed enhanced antimicrobial activity against MRSA and VRSA strains, with an MIC of 4 μg/mL.Substituting hydrogen with halogens such as fluorine 17a increases the antimicrobial activity against MSSA, MRSA and VRSA resistance phenotypes, while introduction of chlorine 18a at the R 1 position weakens the antimicrobial activity against all tested S. aureus resistance phenotypes.Intriguingly, adding a nitro group to the R 1 position resulted in compound 19a, which contained three nitro groups and displayed similar MIC profiles against MSSA and MRSA strains (MIC 4 μg/mL), while the VRSA strain exhibited a slightly elevated MIC (8 μg/mL) (Table 3).
Replacing the acidic nitro group at the R 2 position with a basic dimethylamine group resulted in compounds with similar activity.Dimethylamine derivatives 20a and 21a, containing H or F substituents at the R 1 position, displayed comparable activity against all strains.The introduction of Cl at the R 1 position 22a resulted in decreased activity against all strains (MIC 64 μg/mL), while Cl substitution with a nitro group 23a restored activity against all resistance phenotypes (Table 3).Bis(thiazol-5-yl)phenylmethanes containing a naphthalene core and substitutions at R 1 and R 2 were also examined.Compound 24b, containing R 1 -Cl and R 2 -F, exhibited selectively lower MIC against MRSA and VRSA strains (MIC 4 μg/mL) compared to MSSA strains (MIC 8 μg/mL).Replacing the fluorine atom at R 2 with chlorine or bromine (25b and 26b) while maintaining hydrogen at R 1 decreased antimicrobial activity, while the addition of a nitro substituent at R 2 (27-29b) modulated antimicrobial activity based on the R 1 substituent.Compound 27b (R 1 -H; R 2 -NO 2 ) displayed good activity against MRSA and VRSA strains (MIC 4 μg/mL) with the exception of S. aureus SA-1001 (MIC 8 μg/mL), while incorporating fluorine at the R 1 position in compound 28b resulted in enhanced antimicrobial activity against all strains, with an MIC of 2 μg/mL.The presence of a fluorine atom at the R 1 position appeared to be crucial for potent activity, as replacing fluorine with bromine (29b) resulted in considerable decreased antimicrobial activity (Table 2).
Finally, the addition of a dimethylamino substituent at R 2 (30b, R 1 -H) led to stronger activity against MRSA and VRSA phenotypes (MIC 2 μg/mL) compared to MSSA strains (MIC 4 μg/mL), while the addition of fluorine at the R 1 position (31b) further reduced activity against MSSA strains (MIC 8 μg/mL).Based on the results generated from screening, two compounds (23a and 28b) were selected for further studies.The compounds were selected based on their fixed MIC values across various strains with defined resistance profiles.Furthermore, compounds 23a and 28b were selected for further characterization based on their bactericidal activity, as observed in measuring minimal bactericidal concentration (MBC), that was significantly close to MIC values for compounds 23a and 28b (S1 Table in S1 File).
Following the identification of the most potent bactericidal candidates, we examined the effect of serum addition to the antimicrobial activity of compounds 23a and 28b.We conducted in vitro MIC determination in the presence of 50% fetal bovine serum (FBS), to elucidate the serum protein binding properties, crucial for further hit to lead optimization.Compound 23a exhibited a capacity to bind serum proteins, as its MIC was significantly increased (p < 0.005) (MIC 64 μg/ mL) in all tested strains compared to the serum-free control (MIC 4 μg/mL) (Table 4).In contrast, the antimicrobial activity of naphthalene derivative 28b increased the MIC for S. aureus SA-1001 (MSSA) and S. aureus VR13 (VRSA) strains (MIC 4 μg/mL) (Table 4).
These data highlight the robust antimicrobial activity of bis(thiazol-5-yl)phenylmethanes against S. aureus, even in the presence of genetically defined and challenging resistance mechanisms.Additionally, our structure-activity relationship studies revealed that the presence of a naphthalene substitution with fluorine at the R 1 position and a nitro group at the R 2 position is pivotal for eliciting potent antimicrobial activity against S. aureus, while maintaining minimal serum protein binding.

Bis(thiazol-5-yl)phenylmethane derivatives exhibit the ability to disperse mature S. aureus biofilms in vitro
After identifying the two most potent compounds, 23a and 28b, we proceeded to investigate whether bis(thiazol-5-yl)phenylmethanes 23a and 28b could impact S. aureus biofilms, which are known to be crucial for S. aureus virulence and pathogenesis.We compared the anti-biofilm activity of these compounds with that of vancomycin (VAN), a high molecular mass approved antibiotic commonly employed for treating infections caused by Gram-positive pathogens (Fig 2A and 2B).
The compounds 23a and 28b exhibited a concentration-dependent anti-biofilm activity that was evident across all tested strains.Importantly, both compounds displayed significant anti-biofilm activity against all tested S. aureus resistance phenotypes.Specifically, bis(thiazol-5-yl)phenylmethane 23a demonstrated robust antibiofilm activity against MSSA, MRSA, and vancomycin-resistant S. aureus (VRSA) strains.Compound 28b, on the other hand, exhibited remarkably potent antibiofilm activity against all tested strains, surpassing the efficacy of vancomycin (Fig 2A and 2B).
These findings strongly indicate that compounds 23a and 28b possess the capability to exert promising antibiofilm activity against mature S. aureus biofilms in vitro.The notably enhanced anti-biofilm activity of compound 28b implies the crucial role played by the fluorine and nitro substituents in conferring this biofilm-disrupting capability.

Bis(thiazol-5-yl)phenylmethane derivatives 23a and 28b interacts with S. aureus MurC protein
After performing in silico docking analysis using compounds 23a and 28b and twelve S. aureus Mur ligases, the compounds 23a and 28b were calculated to bind more strongly to MurC with ΔG bin values of -9.6 and -11.4 respectively, as shown in Table 5.
After performing the initial calculations and identifying S. aureus MurC as a target for compounds 23a and 28b, we then explored potential binding sites and poses of the compounds 23a and 28b into MurC.We found that compound 23a positions in MurC active center and interacts with MurC Lys 10, Ser12 and Arg375 amino acids (Fig 3A and 3B).On the other hand, compound 28b interacts with Ser12, Asn68, Arg347 and Arg375 of MurC by forming hydrogen bonds (Fig 4A and 4B).
After characterizing the interaction positions and binding of compounds 23a and 28b to MurC, we then explored conducted a comparative analysis of 23a and 28b into MurC ligase with t known ligand UDP-N-acetyl-alpha-D-muramate (Fig 5).The molecular docking simulations elucidate the binding affinities of compounds 23a and 28b with S. aureus MurC in comparison to its endogenous substrate, UDP-N-acetyl-alpha-Dmuramate.Evidently, both 23a and 28b exhibit substantial binding properties, with compound 28b demonstrating superior efficacy (ΔGbin = -11.4kcal/mol) relative to compound 23a (ΔGbin = -9.6 kcal/mol).Noteworthy is the formation of hydrogen bonds by both compounds with pivotal residues within the MurC binding site, specifically Ser12 and Arg375, indicating consequential interactions essential for complex stability.Compound 28b further establishes hydrogen bonds with Asn68 and Arg347.The substrate engages in hydrogen bonding interactions with Ser12, Thr113, Cys152, Asp174, Arg298, Tyr313, His343, and Thr344 (Table 6).

Bis(thiazol-5-yl)phenylmethane derivatives demonstrate favorable in vitro cytotoxicity parameters
After demonstrating the potent antimicrobial and anti-biofilm activity of bis(thiazol-5-yl)phenylmethane derivatives, our investigation extended to understanding the cytotoxicity profiles of compounds 10-31.To assess the cytotoxic properties of these compounds against different cell types, we employed A549 human lung adenocarcinoma cell lines to evaluate their effects on epithelial cells.Additionally, we utilized the THP-1 monocyte-derived macrophage model to assess cytotoxicity to phagocytic and immune cells (Fig 6).
The bis(thiazol-5-yl)phenylmethane derivatives 10-31 exhibited a structure-dependent cytotoxic activity against both A549 human lung adenocarcinoma cells and THP-1 monocytederived macrophages in vitro.Notably, the cytotoxic properties of R 2 fluorinated compounds (10-12)a were influenced by the substituent at the R 1 position.For instance, compound 10a (R 1 -H) significantly reduced the viability of A549 cells and THP-1 macrophages to 63.4% and  In contrast, compounds bearing naphthalene substitutions (24-31)b demonstrated low cytotoxicity profiles in both cell culture models, reducing the viability of cells to between 75% and 93%.Following the characterization of the in vitro cytotoxic properties of compounds 10-31, we proceeded to assess the cytotoxic and hemolytic properties of the most promising antimicrobial hit compounds, 23a and 28b, using a rabbit erythrocyte hemolysis assay.In this assay, rabbit erythrocytes exposed to 100 μM of compounds 23a and 28b for 1 hour at 37˚C displayed significant hemolysis (p<0.05)compared to the vehicle (DMSO) control.However, it's important to note that none of the compounds exhibited greater or equal hemolysis comparable to the 10% SDS control (Fig 7).
These data demonstrated that bis(thiazol-5-yl)phenylmethane derivatives 10-31 exhibit favorable in vitro cytotoxic profiles crucial for further hit to lead optimization.compounds therefore it is essential to develop novel compounds that are targeting novel bacterial targets, thus evading pre-existing resistance mechanisms [39].This adaptive response contributes to a diminished activity of antimicrobial agents in drug-resistant strains as compared to their pan-susceptible counterparts.
Taking this to the account, we performed further screening of bis(thiazol-5-yl)phenylmethanes using MSSA, MRSA, and VRSA strains.Two bis(thiazol-5-yl)phenylmethane compounds emerged as promising hits, displaying consistent MIC values across diverse S. aureus resistance backgrounds.Specifically, the p-tolyl based derivative 23a, containing nitro and dimethylamino substituents, and the naphthalene based derivative 28b, harboring fluorine and nitro substituents, exhibited notable anti-S.aureus activity against S. aureus isolates with genetically defined resistance phenotypes such as MSSA, MRSA, and VRSA.Furthermore, naphthalene-based derivatives 12, 17 and 19 also showed good antimicrobial activity against S. aureus, although only compounds 23a and 28b showed strong bactericidal activity, with MIC close to minimal bactericidal concentration (MBC).
The cell wall biosynthesis pathway has been extensively explored as a prime target for antimicrobial agents.Numerous investigational and FDA-approved compounds have been introduced to combat infections caused by Gram-positive pathogens.Notably, β-lactam nucleuscontaining antibiotics exemplify this approach by targeting penicillin-binding proteins (PBPs), crucial for peptidoglycan synthesis [35,[38][39][40][41][42].Mutations in PBPs often lead to reduced affinity for β-lactam antibiotics, conferring bacterial resistance.Beyond PBPs, other proteins are pivotal in the cell wall synthesis of Gram-positive bacteria.Within this category, Muramyl ligases (Mur ligases) play a crucial role in peptidoglycan synthesis.The Mur ligase family encompasses distinct enzymes, including MurC, MurD, MurE, MurF, and MurG, each orchestrating specific steps in the peptidoglycan synthesis pathway.Among these, MurC catalyzes the initial addition of the amino acid L-alanine to UDP-N-acetylmuramic acid (UDP-Mur-NAc), a precursor for subsequent cell wall synthesis processes.Inhibition of MurC in S. aureus leads to a loss of bacterial viability and integrity, underscoring the potential of MurC as a promising antimicrobial target [19,35,[38][39][40][41]43].The most promising compounds 23a and 28b were subjected to in silico modeling to propose the cellular target in S. aureus.Among Mur family proteins, MurC was identified as the most promising target with the ability to bind to active center of MurC, thus making bis(thiazol-5-yl)phenylmethanes 23a and 28b as novel S. aureus MurC protein modulators.
Remarkably, when examining serum binding characteristics and performing MIC evaluation using serum, compound 23a exhibited almost complete loss of activity in the presence of serum against tested all strains, whereas compound 28b retained its antimicrobial activity in the serum-containing environment.The p-tolyl substituent is considerably less bulky and hydrophilic, then naphthalene, thus possibly allowing compound 23a to interact more with serum proteins.Furthermore, high lipophilicity mediated by naphthalene substituent in compound 28b could result in stronger interaction with serum lipids thus limiting the protein binding properties.
The assessment of toxicological parameters for novel compounds is pivotal for their subsequent pre-clinical evaluation [43,44].In-depth in vitro toxicity data not only serves as a crucial determinant for guiding hit-to-lead optimization but also offers valuable insights into groups within the compounds that may mediate toxicity.Thiazole derivatives have been extensively investigated as potential anti-cancer candidates, implying that compounds containing the thiazole nucleus may possess inherent cytotoxic properties.In this context, the evaluation of cytotoxic properties of bis(thiazol-5-yl)phenylmethanes was undertaken, utilizing lung epithelial cell models and monocyte-derived macrophages.Surprisingly, the bis(thiazol-5-yl)phenylmethanes exhibited weak cytotoxic activity in both cellular models.To further elucidate the detergent-like cytotoxic activity the most promising compounds (23a and 28b) underwent evaluation using a rabbit erythrocyte lysis assay [43].Both compounds induced lysis of rabbit erythrocytes compared to the vehicle control.However, it is noteworthy that the extent of lysis was significantly lower when compared to the sodium dodecyl sulfate control, a known detergent with strong membrane-disrupting properties.
These results suggest that the cytotoxic activity of bis(thiazol-5-yl)phenylmethanes, although present, may not be predominantly mediated by detergent-like mechanisms [43].The comprehensive evaluation of their cytotoxicity across different cell models and the confirmation of a milder erythrocyte lysis profile, as compared to the control, emphasize the need for a nuanced understanding of the mechanisms underlying the observed cytotoxic properties.Further investigations into the specific cellular pathways impacted by these compounds and their potential therapeutic implications will be essential for their progression in pre-clinical development.
This study provides structure-activity relation information on novel class of bis(thiazol-5-yl)phenylmethanes and sheds some light on their future evaluation as antimicrobials or antibiofilm candidates with activity spanning across different resistance phenotypes.Further studies are needed to understand the safety, in vivo activity and pharmacological properties of the most promising bis(thiazol-5-yl)phenylmethanes and their inhibitory properties directed towards S. aureus MurC.

13 C
https://doi.org/10.1371/journal.pone.0300380.t001have already been crystallized in the reaction mixture.The obtained appropriate product was filtered off, washed with water, and dried.The products were elucidated based on their IR, NMR, and mass spectroscopy data.The analysis of the 1 H NMR spectra of compounds (24-31)b revealed a singlet at approx.5.74 ppm, ascribed to the newly formed C-CH-C fragment, which is clearly confirmed by the resonance line at approx.40.8 ppm in the 13 C NMR spectra.Both spectra displayed an increased abundance of the aromatic signals as well.The characteristic resonances lines at 160.99 ppm (J = 255.7 Hz, 13 C NMR spectra) and at 115.54 (J = 21.4Hz, NMR spectra) show the splitting influence of fluorine atom in the compound 24b.

Fig 1 .
Fig 1.In vitro antimicrobial activity of bis(thiazol-5-yl)phenylmethane derivatives 10-31 and control antimicrobial compounds at a concentration of 100 μM against selected multidrug-resistant bacterial strains.Bacterial strains were exposed to compounds and control antimicrobial drugs at a fixed concentration of 100 μM for 18 hours.Subsequently, resazurin (25 μM) was added, and the plates were further incubated for 3 hours.Following incubation, the optical density at 700 nm (OD 700 nm ) was measured, and the post-treatment viability percentage was normalized to the untreated control (UC).AZT-aztreonam, COL-colistin, FOX-cefoxitin, MEM-meropenem, OXoxacillin,VAN-vancomycin.The data presented in the figure represents the mean ± standard deviation (SD) from three independent experimental replicates.https://doi.org/10.1371/journal.pone.0300380.g002

Fig 2 .
Fig 2. Bis(thiazol-5-yl)phenylmethane derivatives 23a and 28b exhibit a anti-biofilm activity against S. aureus with genetically defined resistance mechanisms.Bacterial biofilms were grown for 24 hours in static conditions and then biofilms were exposed with compounds 23a, 28b or vancomycin (VAN) in fresh media for 18 hours.Biofilm mass was measured by using crystal violet assay.Panel A shows representative images of crystal violet assay.Panel B demonstrates the spectrophotometric measurements of compound-exposed biofilms.The data presented in the figure represents the mean ± standard deviation (SD) from three independent experimental replicates.https://doi.org/10.1371/journal.pone.0300380.g003

). Vancomycin-intermediate S. aureus, Clostri- diodes difficile (C. difficile), Candida auris (C. auris) strains were acquired from the Centers for Disease Control and Prevention (CDC) ARisolate bank. Prior to the initiation of this study, all microbial strains were stored in commercial cryopreservation systems at a temperature of
-80˚C.The strains were cultivated on Columbia Sheep Blood agar (Becton Dickinson, United States), Anaerobe agar for C. difficile (Becton Dickinson, United States) or Potato-Dextrose agar (PDA) for C. auris (Becton Dickinson, United States).